Control of epithelial cell differentiation has important applications in tissue engineering, wound healing and medical devices, such as percutaneous devices. Epithelial tissues such as skin and mucosal tissue typically contain a nonviable barrier layer. Such layers provide various essential functions to a mammal, including the retention of water, exclusion of hostile elements of the environment, such as toxins, allergens or pathogens. All external surfaces of the body are lined by epithelial cells, which provide an important barrier function. In the skin, mouth, and esophagus, this barrier is provided by stratified squamous epithelial cells (see Leeson, T. S., and Leeson, C. R., Histology: 4th Ed. Philadelphia, W. B. Sauders (1981)). In these stratified structures, the basal layer contains the stem or progenitor cells. Once these stem cells or progenitor cells are stimulated to enter the differentiation pathway, their protein expression changes and they move toward the surface, eventually to be sloughed off. (Eckert, R. L. et al., Physiol Rev. 77(2):397-424 (1997)). This process provides a constant renewal of the epithelial barrier. For example, the barrier of the skin, stratum corneum, is formed of non-viable anucleate keratinocytes that have undergone a differentiation and apoptotic events to become corneocytes.
In the context of tissue engineering, many organs and tissues require the development of a competent epithelial lining, including, but not limited to skin, esophagus and oral mucosa, however, the molecular events underlying the growth-arrest, terminal differentiation and apoptosis of the keratinocytes and comeogenesis remain elusive (see, e.g., Roop, Science 267:474-75 (1995)). Currently, the most widely studied inducer of keratinocyte differentiation is the calcium ion. (Menon et al., Cell. Tissue Res. 270:503-512 (1992)).
The Notch signaling pathway is important in regulating development, such as cell growth, proliferation, survival, migration and differentiation. Notch is a type I transmembrane receptor that is activated when bound by a transmembrane Notch ligand containing a Delta, Serrate, Lag-s (“DSL”) conserved domain expressed on the surface of an adjacent cell. Currently there are four known mammalian Notch receptors (Notch 1-4) and four mammalian Notch ligands containing a DSL domain (Delta 1, 2 and Jagged 1, 2) (Artavanis-Tsakonas et al., Science 268:225-232 (1995)). The DSL family of Notch ligands all have multiple epidermal growth factor (EGF) regions in their extracellular domains and they all possess a characteristic DSL domain which is required for function. Henderson et al., Development 120:2913-2924 (1994)). The Notch signaling pathway is initiated through direct cell-cell interactions between the Notch receptors and Notch DSL containing ligands. Upon binding of Notch by its ligand, the transmembrane domain of Notch is proteolytically cleaved and translocates to the nucleus where it binds CSL, the universal transcriptional effector of Notch signaling, thereby inducing gene transcription (see Schweisguth, F., Curr. Biol. 14(3):R129-138 (2004)). In addition, there is evidence that other CSL-independent modes of Notch signaling exist and research is ongoing in this area (see Martinez Arias et al., Curr. Opin. Gen. Dev. 12:524-533 (2002)).
Since both the Notch ligand and Notch receptor are transmembrane proteins, it is generally believed that direct cell-cell interactions are required for activating the Notch signaling pathway. Notch signaling is implicated in many developmental processes in a variety of cell types. It has been shown that Jagged-Notch signaling specifies cell fate, regulates pattern formation, defines boundaries between different cell types, and modulates cell development of the vasculature (see Shimizu, K. et al., J. Biol. Chem. 274:32961 (1999)). It has further been shown that soluble, non-transmembrane forms of Jagged 1 are capable of maintaining the survival and enhance the expansion of human stem cells that are capable of reconstituting hematopoietic lineages in vivo (see Karanu, F. et al., J. Exp. Med 192:1365 (2000)). However, depending on the cell types involved and how the soluble forms of the Notch ligand are presented, Notch ligand binding can result in either activation or inhibition of Notch signaling. (See Hicks C. et al., J. Neurosci. Res. 68:655 (2002)).
Research has been previously performed to evaluate the effect of Notch signaling on stratified squamous epithelial cells, however, previous studies have presented Notch ligands as either soluble proteins or expressed on the surface of adjacent cells. Depending on how soluble forms of the Notch ligand are presented, ligand binding can result in activation or inhibition of Notch signaling (Hicks et al., J. Neurosci. Res. 68:655 (2002)).